Cellular life span and the Warburg effect

Enhanced glycolysis is observed in most of cancerous cells and tissues, called as the Warburg effect. Recent advance in senescent biology implicates that the metabolic shift to enhanced glycolysis would be involved in the early stage during multi-step tumorigenesis in vivo. Enhanced glycolysis is essential both in the step of immortalization and transformation, as it renders cells resistant to oxidative stress and adaptive to hypoxic condition, respectively. ES, immortalized primary, and cancerous cells display the common concerted metabolic shift, including enhanced glycolysis with reduced mitochondrial respiration by poorly characterized mechanism. Discovery of a novel regulatory mechanism for such a metabolic shift might be essential for the future development of cancer diagnosis and anti-cancer therapy.     

The Warburg effect and its cancer therapeutic implications

Increased aerobic glycolysis in cancer, a phenomenon known as the Warburg effect, has been observed in various tumor cells and represents a major biochemical alteration associated with malignant transformation. Although the exact molecular mechanisms underlying this metabolic change remain to be elucidated, the profound biochemical alteration in cancer cell energy metabolism provides exciting opportunities for the development of therapeutic strategies to preferentially kill cancer cells by targeting the glycolytic pathway. Several small molecules capable of inhibiting glycolysis in experimental systems have been shown to have promising anticancer activity in vitro and in vivo. This review article provides a brief summary of our current understanding of the Warburg effect, the underlying mechanisms, and its influence on the development of therapeutic strategies for cancer treatment.     

Comparative metabolic flux profiling of melanoma cell lines: beyond the Warburg effect

Metabolic rewiring is an established hallmark of cancer, but the details of this rewiring at a systems level are not well characterized. Here we acquire this insight in a melanoma cell line panel by tracking metabolic flux using isotopically labeled nutrients. Metabolic profiling and flux balance analysis were used to compare normal melanocytes to melanoma cell lines in both normoxic and hypoxic conditions. All melanoma cells exhibited the Warburg phenomenon; they used more glucose and produced more lactate than melanocytes. Other changes were observed in melanoma cells that are not described by the Warburg phenomenon. Hypoxic conditions increased fermentation of glucose to lactate in both melanocytes and melanoma cells (the Pasteur effect). However, metabolism was not strictly glycolytic, as the tricarboxylic acid (TCA) cycle was functional in all melanoma lines, even under hypoxia. Furthermore, glutamine was also a key nutrient providing a substantial anaplerotic contribution to the TCA cycle. In the WM35 melanoma line glutamine was metabolized in the "reverse" (reductive) direction in the TCA cycle, particularly under hypoxia. This reverse flux allowed the melanoma cells to synthesize fatty acids from glutamine while glucose was primarily converted to lactate. Altogether, this study, which is the first comprehensive comparative analysis of metabolism in melanoma cells, provides a foundation for targeting metabolism for therapeutic benefit in melanoma.     

Metabolic flux analysis gives an insight on verapamil induced changes in central metabolism of HL-1 cells

Verapamil has been shown to inhibit glucose transport in several cell types. However, the consequences of this inhibition on central metabolism are not well known. In this study we focused on verapamil induced changes in metabolic fluxes in a murine atrial cell line (HL-1 cells). These cells were adapted to serum free conditions and incubated with 4 μM verapamil and [U-¹³C₅] glutamine. Specific extracellular metabolite uptake/production rates together with mass isotopomer fractions in alanine and glutamate were implemented into a metabolic network model to calculate metabolic flux distributions in the central metabolism. Verapamil decreased specific glucose consumption rate and glycolytic activity by 60%. Although the HL-1 cells show Warburg effect with high lactate production, verapamil treated cells completely stopped lactate production after 24 h while maintaining growth comparable to the untreated cells. Calculated fluxes in TCA cycle reactions as well as NADH/FADH₂ production rates were similar in both treated and untreated cells. This was confirmed by measurement of cell respiration. Reduction of lactate production seems to be the consequence of decreased glucose uptake due to verapamil. In case of tumors, this may have two fold effects; firstly depriving cancer cells of substrate for anaerobic glycolysis on which their growth is dependent; secondly changing pH of the tumor environment, as lactate secretion keeps the pH acidic and facilitates tumor growth. The results shown in this study may partly explain recent observations in which verapamil has been proposed to be a potential anticancer agent. Moreover, in biotechnological production using cell lines, verapamil may be used to reduce glucose uptake and lactate secretion thereby increasing protein production without introduction of genetic modifications and application of more complicated fed-batch processes.     

Mathematical modelling of the Warburg effect in tumour cords

The model proposed here links together two approaches to describe tumours: a continuous medium to describe the movement and the mechanical properties of the tissue, and a population dynamics approach to represent internal genetic inhomogeneity and instability of the tumour. In this way one can build models which cover several stages of tumour progression. In this paper we focus on describing transition from aerobic to purely glycolytic metabolism (the Warburg effect) in tumour cords. From the mathematical point of view this model leads to a free boundary problem where domains in contact are characterized by different sets of equations. Accurate stitching of the solution was possible with a modified ghost fluid method. Growth and death of the cells and uptake of the nutrients are related through ATP production and energy costs of the cellular processes. In the framework of the bi-population model this allowed to keep the number of model parameters relatively small.     

Lactate stimulates fibroblast expression of hyaluronan and CD44: the Warburg effect revisited

Hyaluronan, a high-molecular-weight glycosaminoglycan of the extracellular matrix, is prominent during rapid tissue growth and repair. It stimulates cell motility and hydrates tissue, providing an environment that facilitates cell movement. Markedly enhanced levels of hyaluronan also occur in the stroma surrounding human cancers, thus providing an environment that promotes spread of cancer cells. The ability of malignant tumors to generate lactate, even in the presence of adequate oxygen, is known as the Warburg effect. Early in wound healing as blood and oxygen supply decrease, lactate levels increase, as does stromal hyaluronan, suggesting a cause-and-effect relationship. Similarly, peritumor stromal fibroblast hyaluronan may be a response to cancer cell lactate. To test this, fibroblasts were cultured in the presence of lactate. With increasing lactate, higher levels of hyaluronan were observed, as were levels of CD44 expression, the predominant receptor for hyaluronan. The ability of tumor cells to utilize anaerobic metabolism and to generate lactate, even in the presence of adequate supplies of oxygen, may be one of the mechanisms used to recruit host fibroblasts to deposit hyaluronan and to express CD44, thereby participating in the process of cancer invasion and metastasis.     

The unveiling of the Warburg effect and the inscribed innovative approach to a radical non toxic anticancer therapy

The purpose of this research has been deciphering the Warburg paradox, the biochemical enigma unsolved since 1923. We solved it by demonstrating that its specific character, i.e. the forced aerobic lactate exportation, represents a crucial metabolic device to counteract the cytotoxic effect produced by an excess of pyruvate at the connection of glycolysis with the Krebs cycle. This solution was verified by exposing cancer cells of different histogenesis to pyruvate concentrations higher than the physiological ones, after showing that these concentrations are totally innocuous when injected into mice. The mechanism of the pyruvate cytotoxicity relies on the saturation of the respiratory chain, leading to a negative shift of the cytosolic NADP/NADPH ratio and the consequent restriction of the purine synthesis and the related cell apoptosis. The reducing equivalents generated by glycolysis and by cytosolic metabolism compete each other for their disposal trough the respiratory chain; this makes it that the cytotoxicity of pyruvate is inversely related to the mitochondrial number and efficiency of various cell types. Thus, the cytotoxicity is high in anaplastic cancer stem cells, whose mitochondria are extremely few and immature (cristae-poor); on the contrary, no inhibition is brought about in adult differentiated cells, physiologically rich of mature mitochondria. All this generates the pyruvate anticancer selectivity, together with the lack of a general toxicity, making pyruvate represent an ideal candidate for a radical non toxical anticancer treatment.     

乏氧微环境、“瓦伯格”效应及上皮间质转化相关性

肿瘤细胞在氧气充足的情况下以糖酵解的方式供能,这一现象称为“瓦伯格”效应,被认为是肿瘤的第七大特征。上皮间质转化（epithelial mesenchymal transition,EMT）是一种重要的细胞过程,参与胚胎发育、伤口愈合及肿瘤的发生等过程中,被认为是恶性肿瘤的重要特征。近年研究表明,“瓦伯格”效应和上皮间质转化的发生均与肿瘤处于乏氧微环境密切相关。乏氧微环境除可直接诱导上皮间质转化发生外,还可诱导肿瘤细胞产生“瓦伯格”效应,进一步促进上皮间质转化的发生。本文就乏氧微环境、“瓦伯格”效应、以及上皮间质转化的相关性的研究进展做一综述,有助于揭示乏氧微环境、肿瘤能量代谢改变以及肿瘤迁移侵袭之间的因果关联。     

Gerometabolites: The pseudohypoxic aging side of cancer oncometabolites

Oncometabolites are defined as small-molecule components (or enantiomers) of normal metabolism whose accumulation causes signaling dysregulation to establish a milieu that initiates carcinogenesis. In a similar manner, we propose the term “gerometabolites” to refer to small-molecule components of normal metabolism whose depletion causes signaling dysregulation to establish a milieu that drives aging. In an investigation of the pathogenic activities of the currently recognized oncometabolites R(-)-2-hydroxyglutarate (2-HG), fumarate, and succinate, which accumulate due to mutations in isocitrate dehydrogenases (IDH), fumarate hydratase (FH), and succinate dehydrogenase (SDH), respectively, we illustrate the fact that metabolic pseudohypoxia, the accumulation of hypoxia-inducible factor (HIFα) under normoxic conditions, and the subsequent Warburg-like reprogramming that shifts glucose metabolism from the oxidative pathway to aerobic glycolysis are the same mechanisms through which the decline of the “gerometabolite” nicotinamide adenine dinucleotide (NAD)⁺ reversibly disrupts nuclear–mitochondrial communication and contributes to the decline in mitochondrial function with age. From an evolutionary perspective, it is reasonable to view NAD⁺-driven mitochondrial homeostasis as a conserved response to changes in energy supplies and oxygen levels. Similarly, the natural ability of 2-HG to significantly alter epigenetics might reflect an evolutionarily ancient role of certain metabolites to signal for elevated glutamine/glutamate metabolism and/or oxygen deficiency. However, when chronically altered, these responses become conserved causes of aging and cancer. Because HIFα-driven pseudohypoxia might drive the overproduction of 2-HG, the intriguing possibility exists that the decline of gerometabolites such as NAD⁺ could promote the chronic accumulation of oncometabolites in normal cells during aging. If the sole activation of a Warburg-like metabolic reprogramming in normal tissues might be able to significantly increase the endogenous production of bona fide etiological determinants in cancer, such as oncometabolites, this undesirable trade-off between mitochondrial dysfunction and activation of oncometabolites production might then pave the way for the epigenetic initiation of carcinogenesis in a strictly metabolic-dependent manner. Perhaps it is time to definitely adopt the view that aging and aging diseases including cancer are governed by a pivotal regulatory role of metabolic reprogramming in cell fate decisions.     

The bicarbonate effect,oxygen evolution,and the shadow of Otto Warburg

A short list of the twentieth century's dominant figures in photosynthesis would unquestionably include Otto Warburg. One of his many discoveries, the `bicarbonate effect' remains a lasting puzzle to his heirs in the field. Recent developments in this area of research have renewed interest and call for a re-examination of the ideas surrounding this controversial topic. Focus here will be on hypotheses developed by a small number of researchers who proposed that bicarbonate may be involved in oxygen evolution. The effect of bicarbonate on the acceptor side of Photosystem II (PS II) is discussed by Jack van Rensen (in this issue). This revised version was published online in June 2006 with corrections to the Cover Date.     

Phosphate and succinate use different mechanisms to inhibit sugar-induced cell death in yeast: insight into the Crabtree effect

Stationary-phase Saccharomyces cerevisiae cells transferred from spent rich media into water live for weeks, whereas the same cells die within hours if transferred into water with 2% glucose in a process called sugar-induced cell death (SICD). Our hypothesis is that SICD is due to a dysregulated Crabtree effect, which is the phenomenon whereby glucose transiently inhibits respiration and ATP synthesis. We found that stationary-phase cells in glucose/water consume 21 times more O(2) per cell than exponential-phase cells in rich media, and such excessive O(2) consumption causes reactive oxygen species to accumulate. We also found that inorganic phosphate and succinate protect against SICD but by different mechanisms. Phosphate protects by triggering the synthesis of Fru-1,6-P(2), which inhibits respiration in isolated mitochondria. Succinate protects in wild-type cells but fails to protect in dic1Δ cells. DIC1 codes for a mitochondrial inner membrane protein that exchanges cytosolic succinate for matrix phosphate. We propose that succinate depletes matrix phosphate, which in turn inhibits respiration and ATP synthesis. In sum, restoring the Crabtree effect, whether with phosphate or succinate, protects cells from SICD.     

AKT-mediated phosphorylation of ATG4B impairs mitochondrial activity and enhances the Warburg effect in hepatocellular carcinoma cells

Phosphorylation is a major type of post-translational modification, which can influence the cellular physiological function. ATG4B, a key macroautophagy/autophagy-related protein, has a potential effect on the survival of tumor cells. However, the role of ATG4B phosphorylation in cancers is still unknown. In this study, we identified a novel phosphorylation site at Ser34 of ATG4B induced by AKT in HCC cells. The phosphorylation of ATG4B at Ser34 had little effect on autophagic flux, but promoted the Warburg effect including the increase of L-lactate production and glucose consumption, and the decrease of oxygen consumption in HCC cells. The Ser34 phosphorylation of ATG4B also contributed to the impairment of mitochondrial activity including the inhibition of F₁Fo-ATP synthase activity and the elevation of mitochondrial ROS in HCC cells. Moreover, the phosphorylation of ATG4B at Ser34 enhanced its mitochondrial location and the subsequent colocalization with F₁Fo-ATP synthase in HCC cells. Furthermore, recombinant human ATG4B protein suppressed the activity of F₁Fo-ATP synthase in MgATP submitochondrial particles from patient-derived HCC tissues in vitro. In brief, our results demonstrate for the first time that the phosphorylation of ATG4B at Ser34 participates in the metabolic reprogramming of HCC cells via repressing mitochondrial function, which possibly results from the Ser34 phosphorylation-induced mitochondrial enrichment of ATG4B and the subsequent inhibition of F₁Fo-ATP synthase activity. Our findings reveal a noncanonical working pattern of ATG4B under pathological conditions, which may provide a scientific basis for developing novel strategies for HCC treatment by targeting ATG4B and its Ser34 phosphorylation.     

The metabolic microenvironment of melanomas: Prognostic value of MCT1 and MCT4

BRAF mutations are known drivers of melanoma development and, recently, were also described as players in the Warburg effect, while this reprogramming of energy metabolism has been identified as a possible strategy for treating melanoma patients. Therefore, the aim of this work was to evaluate the expression and prognostic value of a panel of glycolytic metabolism-related proteins in a series of melanomas. The immunohistochemical expression of MCT1, MCT4, GLUT1, and CAIX was evaluated in 356 patients presenting melanoma and 20 patients presenting benign nevi. Samples included 20 benign nevi, 282 primary melanomas, 117 lymph node and 54 distant metastases samples. BRAF mutation was observed in 29/92 (31.5%) melanoma patients and 17/20 (85%) benign nevi samples. NRAS mutation was observed in 4/36 (11.1%) melanoma patients and 1/19 (5.3%) benign nevi samples. MCT4 and GLUT1 expression was significantly increased in metastatic samples, and MCT1, MCT4 and GLUT1 were significantly associated with poor prognostic variables. Importantly, MCT1 and MCT4 were associated with shorter overall survival. In conclusion, the present study brings new insights on metabolic aspects of melanoma, paving the way for the development of new-targeted therapies.